Epitranscriptomic modifications of coding and non-coding RNAs are emerging as a further layer of gene expression regulation. In this project I will focus on the role of 5-methyl-cytosine (5mC) and 5-hydroxy-methyl-cytosine (5hmC) modifications in mature human microRNAs. I have collected preliminary results by bisulfite NGS analysis of microRNAs in different human cell lines and primary tissues which highlight widespread 5(h)mC modification of mature microRNAs. Furthermore, I have confirmed such modification by dot-blot and RNA immunoprecipitation followed by qPCR using specific antibodies raised against 5mC and 5hmC. I will assess the role of these modifications on microRNA function by assessing subcellular localization, pre-miRNA processing into mature miRNA, mature miRNA stability and target mRNA repression by 5mC and 5hmC modified microRNAs. I will focus this analysis on two 5(h)mC modified, cancer related microRNAs (mir-16-5p and miR-34a-5p). Furthermore, I will identify writer enzymes of these modifications by looking at global microRNA 5(h)mC profile by bisulfite NGS and at single miRNA modifications by RIP in loss-of-function cellular models. This project will yield molecular characterization of the mechanisms through which these modifications are deposed and a mechanistic explanation of their effect on microRNA activity possibly unraveling links with pathological and physiological processes. Furthermore, these data will set the basis for the therapeutic use of 5(h)mC modified miRNA mimics.
Feasibility of my project is supported by several preliminary data collected by my lab in the last couple of years. Briefly, 5mC deposition in miRNAs is supported by NGS of the bisulfite-treated small RNA fraction (BS-sRNA-Seq) human cell lines (HeLaS3, HEK293T, Figure 1) and Peripheral Blood Mononucleated Cells from two healthy donors . Importantly, murine bisulfite RNA-seq datasets retrieved from SRA repository corroborated these findings (data not shown).
By using specific antibodies raised against 5hmC, we highlighted by dot blot analysis that small RNAs (18-50 nt long) harbour 5hmC in HeLaS3 and HEK293T cells (data not shown). Finally, RIP with anti 5mC and anti-5hmC antibodies followed by qRT-PCR of single miRNA validated 5mC and 5hmC modification in HeLaS3 cells.
We started collecting preliminary data on the role of 5mC in microRNA biology focusing on miR-34a-5p. Indeed, our data suggest that transfection of a miR-34a-5p mimic harboring a 5mC at position 4 (5mC-miR-34a-5p) results into a more pronounced effect on previously validated direct (BCL2, ZNF281 and HDAC1) and indirect (p21, whose induction is triggered by miR-34a by promoting p53 transcriptional activity (Yamakuchi, Ferlito, and Lowenstein 2008)) targets in HCT116 cells (Figure 2), one representative replicate of two). This result is in contrast with the one obtained for other miRNAs by Cheray et al (Cheray et al. 2020) in other model systems. However such discrepancy might be explained by different cell systems (e.g. 5mC modified might undergo different further processing to 5hmC/5camC in different cell lines), by the fact that 5mC exerts different effects on different miRNAs or by other factors. Indeed epitranscriptomic modifications may generally exert a substrate specific effect depending on sequence context and/or on the reader protein.
Furthermore, our data not only prove that 5mC is a widespread miRNA modification, but also support the feasibility of high-throughput profiling with NGS based techniques and validation through RIP and qRT-PCR (Carissimi et al, manuscript in preparation).
All in vitro experiments will be performed in four independent biological replicates.
The main outcomes expected by the project Aims are the following:
Aim 1: Assessment of the role of 5(h)mC in microRNA biology. Understanding how 5(h)mC modification affects microRNA activity and/or processing and hence the expression of their targets is a fundamental achievement to unravel the contribution of this layer of regulation to the overall role of miRNA in post-transcriptional gene expression regulation. Furthermore these data might provide important information which might drive the design of 5(h)mC modified therapeutic mimic miRNAs.
Aim 2: the enzymes responsible for 5(h)mC modification of microRNAs. KD cell lines in which microRNA 5(h)mC modification is significantly impaired will be further exploited to correlate loss of microRNA 5(h)mC with gene expression regulation by microRNAs of selected miRNA targets.